The Flavivirus genus includes mosquito-borne human pathogens, such as dengue virus, yellow fever virus, and the Zika virus (ZIKV), among others. ZIKV's recent spread in Americas and its link to microcephaly in newborn infants and the Guillan-Barr syndrome in adults has invigorated efforts to develop a vaccine, as well as measures to eradicate the Aedes mosquito vectors. In addition to these measures, it is equally important to develop antivirals based on targeting enzymatic activities central to the life cycle and survival of ZIKV. One such enzymatic activity is encoded by the methyltransferase (MTase) domain, located at the N-terminus of the nonstructural protein NS5. As a step towards this goal, we have determined several high-resolution crystal structures of the ZIKV NS5-MTase. Together, these structures (all determined to better than 1.5 in resolution) provide a powerful new framework for the design and synthesis of small molecules that selectively target MTase from ZIKV and other pathogenic flaviviruses. In aim 1, we will a) use the structural information to design S-adenosylmethionine (SAM) based analogs with the capacity to interact with unique amino acids in ZIKV MTase, and which extend deeper into the RNA binding tunnel to block RNA binding. We will also design analogs with substituents on both the adenine base and the methionine portion of SAM to provide additional selectivity against the human RNA 5'-cap MTases. We will b) chemically synthesize the analogs and produce them to a purity of 95% for in vitro and cell-based assays, as well as for cocrystallization with the ZIKV NS5- MTase. In aim 2, we will a) perform biophysical assays to assess the ability of these analogs to selectively bind the ZIKV NS5-MTase as compared to the human RNA 5'-cap MTases, and test their ability to inhibit RNA methylation; b) test these analogs in viral cell based assays to assess their efficacy in blocking viral replication; c) determine structures ZIKV NS5-Mtase with select analogs for additional, iterative rounds of structure activity relationships (SARs). Together, the proposed studies will help to identify new small molecules that can be potentially developed into inhibitors of ZIKV NS5-MTase.